(1) Field of the Invention
This invention relates to the field of specific binding assays and reagents therefor, particularly those in which some of the reagents are bound to a solid phase and used to determine ligands in liquid samples containing interfering substances.
(2) Brief Description of the Prior Art
The development of specific binding assay techniques has provided extremely useful analytical methods for determining various organic substances of diagnostic, medical, environmental and industrial importance which appear in liquid media at very low concentrations. Specific binding assays are based on the specific interaction between a ligand, i.e., a bindable analyte under determination, and a binding partner therefor, i.e., receptor. Where one of the ligand and its binding partner is an antibody and the other is a corresponding hapten or antigen, the assay is known as an immunoassay.
These specific binding assays have been provided in a variety of solid state formats including analytical elements or test strips, coated tubes, particle-associated reagents and others. Agglutination assays are among the most widely used solid state specific binding assays, usually as immunoassays. They may be classified as direct, indirect (passive) or inhibition type agglutination assays. In a direct agglutination assay, particles having surface components which are one member of a specific binding pair (e.g., a receptor), are reacted with a sample to be assayed for the other member of the specific binding pair (e.g., ligand). In the indirect (passive) agglutination format, one member of a specific binding pair (e.g., receptor) is bound to a solid substrate particle, and this particle-bound member is reacted with a sample to be assayed for the other member of the pair (e.g., ligand). In inhibition-type agglutination assays, a sample to be tested for one binding pair member is reacted with a solution containing the other member of the binding pair and particles which contain (direct) or are bound with (indirect) the binding pair member suspected of being in the sample. Agglutination assays have been summarized in the literature. See, for example, Bellanti, Immunology, W. B. Saunders Co., Philadelphia (1971), pgs. 139 et seq; and Fudenberg, et al, Basic & Clinical Immunology, Lange Medical Publications, Los Altos, CA. (1976), pp. 308 et seq. Also, Sawai et al, U.S. Pat. Nos. 4,118,192 and 4,208,185 relate to agglutination assays. Earlier references which are likewise relevant are Singer et al, J. Colloid and Interface Science, 45:608-614 (1973) and Faure et al, Protides of the Biological Fluids, Proceedings of the Colloquium, 20:589-593 (1972). A number of agglutination assay test kits for specific analytes or ligands are commercially available and have also been described in the literature. See, for example, Rose, et al (Eds.), Manual of Clinical Immunology, American Society for Microbiology, Washington, D.C. (1978).
When assaying complex liquids, such as human serum, some proteins and other substances can cause non-specific interferences with the agglutination reaction. Therefore, these proteins must be destroyed or their non-specific interaction with the reagents overcome to obtain accurate measurement of ligand concentrations. The most common approach to avoiding non-specific protein interference has been by first digesting the proteins with a proteolytic enzyme such as pepsin. The enzyme is then inactivated or destroyed prior to the assay. For example, Collet-Cassart, et al Clin. Chem., 27:1205-09 (1981) disclose a particle-counting immunoassay for digoxin in samples which were predigested with pepsin. The digestion was stopped by adding tris (hydroxymethyl) methylamine which inactivates the pepsin. See also Chau et al, J. Clin. Endocrinol. Metab., 42:189-192 (1976).
Alternatively, these assays can be performed using particles which are not susceptible to interaction with substances that cause non-specific interference. For example, Hosaka, et al, EPO Application No. 54,249 discloses immunoassay reagents formed of an immunochemical containing an amino group, e.g. an antibody, which is covalently bound to an epoxy group on the surface of particles comprising a polymer having the repeating unit of glycidyl acrylate and/or glycidyl methacrylate. The particles are not to have other hydrophobic components on their surface. When the epoxy groups are not all consumed by binding with the above immunochemicals, hydrophilic proteins, e.g. albumin, which do not interfere with the immunoassay can be reacted with the remaining epoxy groups. It is said that these particles are unlikely to agglutinate non-specifically and are free from non-specific adhesion to cells.